Identification of isoaspartyl-containing sequences in peptides and proteins that are usually poor substrates for the class II protein carboxyl methyltransferase.
We have found that a chicken egg lysozyme derivative (beta-101-lysozyme) containing an L-isoaspartyl residue at position 101 has a Km for methylation by the human erythrocyte L-isoaspartyl/D-aspartyl protein methyltransferase (EC 2.1.1.77) of 183 microM, about 30 times higher than that expected from previous studies with isoaspartyl-containing peptides. In the course of investigating the reasons for this poor enzyme recognition, we found that charged residues on the carboxyl side of isoaspartyl residues had a large effect on the affinity of the enzyme for synthetic peptides. This is best illustrated by the lysozyme-related peptide YVSisoDGDG, which has a Km for methylation of 469 microM. When the penultimate aspartyl residue is replaced by a cysteinyl residue, the Km drops to 4.6 microM, comparable to other peptides of similar size. Furthermore, replacing it with a cysteic acid residue results in a Km of 104 microM, suggesting that a negative charge at this position may lead to a weaker affinity of the peptide substrate for the methyltransferase. Assays with additional synthetic peptides indicate that moving the negative charge to the first or third residue on the carboxyl side of the isoaspartyl residue has a similar but less severe effect in reducing its affinity for the methyltransferase. Enzymatic methylation has recently been proposed to be the first step in the conversion of abnormal isoaspartyl residues to aspartyl residues. The results reported here, however, along with previous evidence that protein tertiary structure can inhibit isoaspartyl methylation, suggest that only a subclass of damaged sites are capable of efficiently entering a putative repair pathway; the sites not recognized by the methyltransferase may accumulate in vivo.
Lowenson
J D
JD
Department of Chemistry and Biochemistry, University of California, Los Angeles 90024.
Clarke
S
S
D000595:Amino Acid Sequence; D001224:Aspartic Acid; D004912:Erythrocytes / Q000201:enzymology; D006801:Humans; D007527:Isoenzymes / Q000097:blood / Q000378:metabolism*; D007536:Isomerism; D008745:Methylation; D008780:Methyltransferases / Q000097:blood / Q000378:metabolism*; D008969:Molecular Sequence Data; D009113:Muramidase; D009842:Oligopeptides / Q000138:chemical synthesis; D010084:Oxidation-Reduction; D011506:Proteins; D013379:Substrate Specificity
D007527:Isoenzymes; D009842:Oligopeptides; D011506:Proteins; D001224:Aspartic Acid; D008780:Methyltransferases; C055685:nicotinate methyltransferase; D009113:Muramidase